Green synthesis of reduced graphene oxide using Nigella sativa  seed extract

ABSTRACT

The green synthesis of reduced graphene oxide nanoparticles using  Nigella sativa  seed extract comprises the steps of mixing a quantity of soot or other carbon source in an acid solution while stirring to obtain a solution; adding a first oxidant gradually into the solution to oxidize the soot and obtain a suspension; stirring the suspension while maintaining the temperature of the suspension at about 35° C.; adding  Nigella sativa  seed extract to the suspension while raising the temperature of the suspension to about 60° C.; adding hydrogen peroxide to the suspension; and isolating the reduced graphene oxide nanoparticles by centrifugation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 15/151,466, filed May 10,2016, now pending.

BACKGROUND OF THE INVENTION

-   -   1. Field of the Invention

The present invention relates to graphene oxide, and particularly to thegreen synthesis of reduced graphene oxide (rGO) using Nigella sativaseed extract and its applications.

-   -   2. Description of the Related Art

One of the recent advances in pharmacological research has been the useof novel drug delivery systems, and also the increasing application ofmonoclonal antibodies and oligonucleotides for therapeutic purpose.Functionalized nano-sized graphene has been used as a drug carrier forin vitro intracellular delivery of anticancer chemotherapy drugs. It hasbeen found that nano-graphene with a biocompatible polyethylene glycol(PEG) coating displays high passive in vivo tumor uptake and could beused for effective photo-thermal ablation of tumors in a mouse model. Onthe other hand, many groups have developed graphene-based biosensors todetect various biomolecules via different mechanisms. Graphene-basednanomedicine, although still in its infancy, appears to be encouragingand may bring novel opportunities for future disease diagnosis andtreatment.

Graphene is an atom-thick monolayer of carbon atoms arranged in a twodimensional honeycomb structure, and it is a basic building block forother graphitic materials, such as graphite and carbon nanotubes.Because of their unique and desirable electrical, optical, mechanicaland chemical characteristics, graphene, graphene oxide (GO), and reducedgraphene oxide (rGO) have been extensively studied for a variety ofapplications, such as nanoelectronics, sensors, energy storage,nanocomposites, etc., including biomedicine. The potential of grapheneas a nanocarrier for drug delivery, gene delivery and nanomedicine hasbeen demonstrated for possible cancer therapies. In addition, theimproved synthesis and versatile surface modification of graphene hasopened up new avenues for research on the nanoscale. In this regard,using “green” methods in the synthesis of nanoparticles has receivedattention, as conventional chemical methods are expensive and requirethe use of hazardous chemical and organic solvents.

Thus, the green synthesis of reduced graphene oxide (rGO) nanoparticlesusing Nigella sativa seed extract solving the aforementioned problems isdesired.

SUMMARY OF THE INVENTION

The green synthesis of reduced graphene oxide nanoparticles usingNigella sativa seed extract comprises the steps of mixing a quantity ofsoot or other carbon source in an acid solution while stirring to obtaina solution; adding a first oxidant gradually into the solution tooxidize the soot and obtain a suspension; stirring the suspension whilemaintaining the temperature of the suspension at about 35° C.; addingNigella sativa seed extract to the suspension while raising thetemperature of the suspension to about 60° C.; adding hydrogen peroxideto the suspension; and isolating the reduced graphene oxidenanoparticles by centrifugation. The method of synthesizing reducedgraphene oxide nanoparticles can further comprise washing the reducedgraphene oxide nanoparticles with deionized water and 5% HCl solutionand drying the product at about 100° C. in an oven.

The reduced graphene oxide nanoparticles can be used in inhibitingcancer cell proliferation. For example, the method of inhibiting thegrowth or proliferation of a cancer cell may comprise the step ofcontacting the cancer cell with an effective amount of the reducedgraphene oxide nanoparticles. The cancer cell can include a breastcarcinoma cell or a colon carcinoma cell.

The reduced graphene oxide nanoparticles can be used in inhibitingmicrobial activity. For example, a method for inhibiting microbialactivity caused by a microorganism comprises the step of administeringan effective amount of the graphene oxide nanoparticles to a site ofmicrobial activity. The microorganisms can be selected from the groupconsisting of yeast, gram positive and gram negative bacteria.

These and other features of the present invention will become readilyapparent upon further review of the following specification anddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the X-ray diffraction pattern of reduced graphene oxideproduced by green synthesis using Nigella sativa seed extract accordingto the present invention.

FIG. 2 is a Zetasizer plot showing particle size distribution of greensynthesized reduced graphene oxide using Nigella sativa seed extractaccording to the present invention.

FIGS. 3A, 3B, and 3C are transmission electron micrographs (TEM) ofgreen synthesized reduced graphene oxide using Nigella sativa seedsextract according to the present invention.

FIGS. 4A and 4B are scanning electron micrographs (SEM) of greensynthesized reduced graphene oxide using Nigella sativa seeds extractaccording to the present invention at different levels of magnification.

FIG. 5 is the Energy Dispersive Spectroscopy spectrum of greensynthesized reduced graphene oxide using Nigella sativa seed extractaccording to the present invention with two dominant peaks for carbonand oxygen atoms, respectively.

FIG. 6 is a plot of cell viability as a function of the concentration ofreduced graphene oxide synthesized from Nigella sativa seeds extractaccording to the present invention for breast carcinoma cells (MCF-7cell line).

FIG. 7 is a plot of cell viability as a function of the concentration ofreduced graphene oxide synthesized from Nigella sativa seeds extractaccording to the present invention for colon carcinoma cells (HCT-116cell line).

Similar reference characters denote corresponding features consistentlythroughout the attached drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The green synthesis of reduced graphene oxide nanoparticles usingNigella sativa seed extract comprises the steps of mixing a quantity ofsoot or other carbon source in an acid solution while stirring to obtaina solution; adding a first oxidant gradually into the solution tooxidize the soot and obtain a suspension; stirring the suspension whilemaintaining the temperature of the suspension at about 35° C.; addingNigella sativa seed extract to the suspension while raising thetemperature of the suspension to about 60° C.; adding hydrogen peroxideto the suspension; and isolating the reduced graphene oxidenanoparticles by centrifugation.

The method of synthesizing reduced graphene oxide nanoparticles canfurther comprise washing the reduced graphene oxide nanoparticles withdeionized water and 5% HCl solution and drying the product at about 100°C. in an oven. The carbon source for producing the reduced grapheneoxide can be soot collected from girdle, which is formed during bakery.The acid can be sulfuric acid. The plant seeds extract used in thesynthesis of reduced graphene oxide nanoparticles are obtained from theplant Nigella sativa. Generally, adding the Nigella sativa seed extractcan be performed at a temperature of the suspension of about 60° C. forabout 15 minutes. Typically, the first oxidant is potassiumpermanganate.

The reduced graphene oxide, which is produced from the synthesis method,are nanoparticles having a mean diameter in the range of from about 5 nmto about 100 nm across the largest dimension. Typically, the grapheneoxide nanoparticles can have one or more shapes selected from the groupconsisting of spherical-shaped, spheroidal-shaped, elongated/sphericalshaped, rod-shaped and faceted shaped.

The reduced graphene oxide nanoparticles can be used in inhibitingcancer cell proliferation. For example, a method of inhibiting thegrowth or proliferation of a cancer cell can comprise the step ofcontacting the cancer cell with an effective amount of the reducedgraphene oxide nanoparticles. The cancer cell can include a breastcarcinoma cell or a colon carcinoma cell.

The reduced graphene oxide nanoparticles can also be used in inhibitingmicrobial activity. For example, a method for inhibiting microbialactivity caused by microorganisms comprises the step of administering aneffective amount of the graphene oxide nanoparticles to a site ofmicrobial activity. The microorganisms can be selected from the groupconsisting of yeast, gram positive and gram negative bacteria.

As used herein the tell “nanoparticle” refers to a particle having atleast one dimension sized between 1 and 100 nanometers. By the term“effective amount”, it is understood that, with respect to, for example,pharmaceuticals, a pharmaceutically effective amount is contemplated. Apharmaceutically effective amount is the amount or quantity of activeingredient that is enough for the required or desired therapeuticresponse, or in other words, the amount that is sufficient to elicit anappreciable biological response when administered to a patient.

As used herein, the term “seed extract” encompasses, for example, anychemical or combination of chemicals found in the seeds of the plant,including the derivatives of the compounds found in the seeds viachemical reaction. The “seed extract” can be obtained from the plant byany process, for example, cold water extraction, hot water extraction,extraction with an organic solvent, and/or extraction with asupercritical solvent. The preferred method of extraction of Nigellasativa seed extract is using boiling water as described below.

Nigella sativa, often called black cumin, is an annual flowering plantin the family Ranunculaceae, native to south and southwest Asia. Nigellasativa grows to 20-30 cm tall, with finely divided, linear leaves.

The present teachings will be understood more readily by reference tothe following examples, which are provided by way of illustration.

Example 1 Preparation of Extract from Nigella Sativa Seeds

Nigella sativa seeds were washed several times with distilled water.Then, about 30 grams of coarsely ground Nigella sativa seeds were takenand boiled in 150 mL of double distilled water for about 5 min. Theextract was centrifuged and then filtered. The filtrate was collectedand stored at 4° C. until further use.

Example 2 Green Synthesis of Reduced Graphene Oxide (rGO) Nanoparticles

Soot was collected from girdle which formed during bakery of Abray, alocal and traditional paste used as drinker in Ramadan month in Sudan.Graphene oxide (GO) was prepared according to the modified Hummermethod. In detail, 1 g of soot was mixed with 50 mL H₂SO₄ and stirredfor 5 min. Next, 5 g of potassium permanganate, KMnO₄, was very slowlyadded in an ice bath. The suspension was again stirred at 35° C. for 10min. The temperature of the mixture was adjusted to a constant 60° C.for 15 min while the Nigella sativa seeds extract was added continuouslyso that the volume of the suspension was 150 mL, and 5 mL of H₂O₂ wasadded after 5 min. The reaction product was centrifuged and washed withdeionized water and 5% HCl solution repeatedly. Finally, the product wasdried at 100° C.

The product was characterized by transmission electron microscopy(JEM-1011, JEOL, Japan). Also, scanning electron microscopy (SEM) wasemployed to characterize the shape and morphologies of font lednanoparticles using JEOL-FE SEM and X-ray diffractometer (XRD), BrukerD8 ADVANCE. The size of the synthesized nanoparticles was analyzedthrough Zetasizer, Nano series, HT Laser, ZEN3600 (Molvern Instrument,UK). JEOL-FE SEM; and Energy Dispersive Spectrometer (EDS) analysis wasperformed for the confirmation of elemental oxygen and carbon. FIG. 1shows the XRD pattern of green synthesized reduced graphene oxide usingNigella sativa seed extract. FIG. 2 shows the particle size distributionof green synthesized reduced graphene oxide using Nigella sativa seedextract. Two peaks having Z-average sizes of about 1.8 nm (peak 1) and300 nm (peak 2) having intensities of about 15% and 30% respectivelywere found. FIGS. 3A-C show the transmission electron microscopy (TEM)images of green synthesized reduced graphene oxide using Nigella sativaseed extract. The particles can be spherical or elongated or rod-like,with a dimension of about 5 nm to 200 nm. FIG. 4 shows the scanningelectron microscopy (SEM) images of green synthesized reduced grapheneoxide using Nigella sativa seed extract. Energy Dispersive Spectrometer(EDS) analysis was performed for the confirmation of elemental oxygenand carbon, as shown in FIG. 5. Table 1 shows the EDS results showingpercentage of elements present in reduced graphene oxide nanoparticlessuspension.

TABLE 1 Elemental analysis of reduced graphene oxide Element Weight %Atomic % C 8.45 10.77 O 87.42 83.66 TOTALS 100.00

Example 3 Antimicrobial Screening

Antimicrobial activity of the reduced graphene oxide (rGO) nanoparticleswas determined using the agar well diffusion assay method as describedby Holder and Boyce, 1994. Three bacterial strains (two gram positiveand one gram negative) and one yeast strain, namely, Bacillis subtilis(RCMB 010067), Staphylococcus pneumoniae (RCMB 010011), Escherichia coli(RCMB 010052), and Aspergillus fumigatus (RCMB 02568) were used for theantimicrobial assay.

The tested organisms were sub-cultured on nutrient agar medium (Oxoidlaboratories, UK) for bacteria and Sabouraud dextrose agar (Oxoidlaboratories, UK) for fungi. Ampicillin and Gentamicin were used aspositive control for gram positive and gram negative bacteria,respectively, while Amphotericin B was used for fungi. The plates weredone in triplicates. Bacterial cultures were incubated at 37° C. for 24h, while the fungal cultures were incubated at 25-30° C. for 3-7 days.Antimicrobial activity was determined by measuring the zone ofinhibition [A. Agwa, (2000)]. Samples were tested at a concentration of100 μl; Data are expressed in the form of (M±S.D.): mean±standarddeviation; Diameter of the inhibition zone (mm) beyond the well diameterof 6 mm; NT: not tested; NA: no activity. Table 2 shows theantimicrobial activity of green synthesized reduced graphene oxidenanoparticles suspension using diffusion agar well diffusion assaymethod.

TABLE 2 Nigella sativa reduced graphene oxide Antibiotic FUNGIAmphotericin B Aspergillus fumigatus 33.3 ± 1.5 21.7 ± 1.5 (RCMB 02567)Candida albicans 19.3 ± 1.5 22.7 ± 1.5 (RCMB 05038) Gram PositiveBacteria: Ampicillin Streptococcus 32.7 ± 2.1 21.0 ± 1.0 pneumoniae(RCMB 010011) Bacillis subtilis (RCMB 34.0 ± 2.0 31.3 ± 1.5 010068) GramnegativeBacteria: Gentamicin Pseudomonas 16.3 ± 2.1 16.0 ± 1.0aeruginosa (RCMB 010045) Escherichia coli 26.3 ± 2.1  20.3 ± 0.58 (RCMB010054)

Example 4 Antitumor Activity Assay

The tested human carcinoma cell lines were obtained from the AmericanType Culture Collection (ATCC, Rockville, Md.). The cells were grown onRPMI-1640 medium supplemented with 10% inactivated fetal calf serum and50 μg/ml gentamycin. The cells were maintained at 37° C. in a humidifiedatmosphere with 5% CO₂ and were subcultured two to three times a week.

For antitumor assays, the tumor cell lines were suspended in medium atconcentration 5×10⁴ cell/well in Corning® 96-well tissue culture platesthen incubated for 24 h. The tested compounds were then added into96-well plates (six replicates) to achieve eight concentrations for eachcompound. Six vehicle controls with media or 0.5% DMSO were run for each96 well plate as a control. After incubating for 24 h, the numbers ofviable cells were determined by the MTT test. Briefly, the media wasremoved from the 96 well plates and replaced with 100 μl of freshculture RPMI 1640 medium without phenol red then 10 μl of the 12 mM MTTstock solution (5 mg of MTT in 1 mL of PBS) to each well including theuntreated controls. The 96 well plates were then incubated at 37° C. and5% CO₂ for 4 hours. An 85 μl aliquot of the media was removed from thewells, and 50 μl of DMSO was added to each well and mixed thoroughlywith the pipette and incubated at 37° C. for 10 min. Then, the opticaldensity was measured at 590 nm with the microplate reader (SunRise,TECAN, Inc., USA) to determine the number of viable cells and thepercentage of viability was calculated using the following equation (1):

$\begin{matrix}{{{Percentage}\mspace{14mu}{of}\mspace{14mu}{Viability}} = {\left\lbrack {1 - \frac{ODt}{ODc}} \right\rbrack \times 100}} & (1)\end{matrix}$

In Equation 1, ODt is the mean optical density of wells treated with thetested sample and ODc is the mean optical density of untreated cells.The relation between surviving cells and drug concentration is plottedto get the survival curve of each tumor cell line after treatment withthe specified compound. The 50% inhibitory concentration (IC₅₀), theconcentration required to cause toxic effects in 50% of intact cells,was estimated from graphic plots of the dose response curve for eachconc. using Graphpad Prism software (San Diego, Calif. USA) (Mosmann,1983; Elaasser et al., 2011). FIG. 6 shows the cytotoxic activity ofreduced graphene Oxide synthesized from Nigella sativa seeds extractsuspension against Breast carcinoma cells (MCF-7 cell line). Table 3shows the inhibitory activity of reduced graphene Oxide synthesized fromNigella sativa extract against breast carcinoma cells was detected underthese experimental conditions with IC₅₀=2.67 μl.

TABLE 3 Standard Sample Viability % (3 Replicates) Inhibition DeviationConc. (μl) 1st 2nd rd Mean % (±) 100 7.92 6.84 7.13 7.30 92.70 0.56 5013.85 14.87 15.41 14.71 85.29 0.79 25 20.42 18.56 22.98 20.65 79.35 2.2212.5 28.93 29.71 31.86 30.17 69.83 1.52 6.25 34.82 37.15 40.72 37.5662.44 2.97 3.125 45.49 46.73 48.24 46.82 53.18 1.38 1.56 57.13 54.8961.82 57.95 42.05 3.54 0.78 74.59 69.27 72.16 72.01 27.99 2.66 0 100 100100 100 0.00

FIG. 7 shows the cytotoxic activity of reduced graphene oxidesynthesized from Nigella sativa seeds extract suspension against coloncarcinoma cells (HCT-116 cell line). Table 4 shows the inhibitoryactivity of reduced graphene Oxide synthesized from Nigella sativaextract against colon carcinoma cells was detected under theseexperimental conditions with IC₅₀=1.41 μl.

TABLE 4 Standard Sample Viability % (3 Replicates) Inhibition Deviationconc. (μl) 1st 2nd 3rd Mean % (±) 100 5.92 6.49 5.38 5.93 94.07 0.56 5012.88 11.36 10.74 11.66 88.34 1.10 25 17.75 16.45 18.52 17.57 82.43 1.0512.5 22.91 23.67 21.74 22.77 77.23 0.97 6.25 26.74 30.68 28.91 28.7871.22 1.97 3.125 32.65 37.94 35.13 35.24 64.76 2.65 1.56 39.87 48.9653.12 47.32 52.68 6.78 0.78 58.72 63.21 60.84 60.92 39.08 2.25 0 100 100100 100 0.00

Thus, the above examples illustrate a simple, non-toxic, cost-effective,quick, and environmentally friendly synthesis approach for the reducedgraphene oxide nanoparticles using Nigella sativa seeds extract. Thepotential of graphene as nanocarriers for drug delivery, gene deliveryand nanomedicine exists for possible cancer therapies. The green methodof synthesizing the reduced graphene oxide nanoparticles can havepotential applications as antibacterial and anti-cancer agents.

It is to be understood that the present invention is not limited to theembodiments described above, but encompasses any and all embodimentswithin the scope of the following claims.

We claim:
 1. Reduced graphene oxide nanoparticles produced according toa method for green synthesis of reduced graphene oxide, the methodcomprising the steps of: a) mixing a quantity of carbon source in anacid while stirring to obtain a solution, b) adding a first oxidantgradually into the solution to oxidize the carbon source and obtain asuspension, c) stirring the suspension while maintaining a temperatureof the suspension at about 35° C., d) adding Nigella sativa seed extractto the suspension while raising the temperature of the suspension toabout 60° C., and e) adding hydrogen peroxide to the suspension; andisolating resulting reduced graphene oxide nanoparticles bycentrifugation, wherein the graphene oxide nanoparticles have a meandiameter between 5 nm and 200 nm across the largest dimension andconsist essentially of the Nigella sativa seed extract.
 2. The reducedgraphene oxide nanoparticles according to claim 1, wherein said grapheneoxide nanoparticles have a shape selected from the group consisting ofspherical-shaped, spheroidal-shaped, elongated/spherical shaped,rod-shaped and faceted shaped.